Subcellular fractionation of stored red blood cells reveals a compartment-based protein carbonylation evolution.

During blood banking, erythrocytes undergo storage lesions, altering or degrading their metabolism, rheological properties, and protein content. Carbonylation is a hallmark of protein oxidative lesions, thus of red blood cell oxidative stress. In order to improve global erythrocyte protein carbonylation assessment, subcellular fractionation has been established, allowing us to work on four different protein populations, namely soluble hemoglobin, hemoglobin-depleted soluble fraction, integral membrane and cytoskeleton membrane protein fractions. Carbonylation in erythrocyte-derived microparticles has also been investigated. Carbonylated proteins were derivatized with 2,4-dinitrophenylhydrazine (2,4-DNPH) and quantified by western blot analyses. In particular, carbonylation in the cytoskeletal membrane fraction increased remarkably between day 29 and day 43 (P<0.01). Moreover, protein carbonylation within microparticles released during storage showed a two-fold increase along the storage period (P<0.01). As a result, carbonylation of cytoplasmic and membrane protein fractions differs along storage, and the present study allows explaining two distinct steps in global erythrocyte protein carbonylation evolution during blood banking. This article is part of a Special Issue entitled: Integrated omics.

Fitness to drive and cannabis: Validation of two blood THCCOOH thresholds to distinguish occasional users from heavy smokers.

Many studies based on either an experimental or an epidemiological approach, have shown that the ability to drive is impaired when the driver is under the influence of cannabis. Baseline performances of heavy users remain impaired even after several weeks of abstinence. Symptoms of cannabis abuse and dependence are generally considered incompatible with safe driving. Recently, it has been shown that traffic safety can be increased by reporting the long-term unfit drivers to the driver licensing authorities and referring the cases for further medical assessment. Evaluation of the frequency of cannabis use is a prerequisite for a reliable medical assessment of the fitness to drive. In a previous paper we advocated the use of two thresholds based on 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) concentration in whole blood to help to distinguish occasional cannabis users (≤3μg/L) from heavy regular smokers (≥40μg/L). These criteria were established on the basis of results obtained in a controlled cannabis smoking study with placebo, carried out with two groups of young male volunteers; the first group was characterized by a heavy use (≥10 joints/month) while the second group was made up of occasional users smoking at most 1 joint/week. However, to date, these cutoffs have not been adequately assessed under real conditions. Their validity can now be evaluated and confirmed with 146 traffic offenders' real cases in which the whole blood cannabinoid concentrations and the frequency of cannabis use are known. The two thresholds were not challenged by the presence of ethanol (40% of cases) and of other therapeutic and illegal drugs (24%). Thus, we propose the following procedure that can be very useful in the Swiss context but also in other countries with similar traffic policies: if the whole blood THCCOOH concentration is higher than 40μg/L, traffic offenders must be directed first and foremost toward medical assessment of their fitness to drive. This evaluation is not recommended if the THCCOOH concentration is lower than 3μg/L and if the self-rated frequency of cannabis use is less than 1 time/week. A THCCOOH level between these two thresholds cannot be reliably interpreted. In such a case, further medical assessment and follow-up of the fitness to drive are also suggested, but with lower priority.

Inflammatory markers and blood pressure: sex differences and the effect of fat mass in the CoLaus Study.

Several studies have reported high levels of inflammatory biomarkers in hypertension, but data coming from the general population are sparse, and sex differences have been little explored. The CoLaus Study is a cross-sectional examination survey in a random sample of 6067 Caucasians aged 35-75 years in Lausanne, Switzerland. Blood pressure (BP) was assessed using a validated oscillometric device. Anthropometric parameters were also measured, including body composition, using electrical bioimpedance. Crude serum levels of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and ultrasensitive C-reactive protein (hsCRP) were positively and IL-1β (IL-1β) negatively (P<0.001 for all values), associated with BP. For IL-6, IL-1β and TNF-α, the association disappeared in multivariable analysis, largely explained by differences in age and body mass index, in particular fat mass. On the contrary, hsCRP remained independently and positively associated with systolic (β (95% confidence interval): 1.15 (0.64; 1.65); P<0.001) and diastolic (0.75 (0.42; 1.08); P<0.001) BP. Relationships of hsCRP, IL-6 and TNF-α with BP tended to be stronger in women than in men, partly related to the difference in fat mass, yet the interaction between sex and IL-6 persisted after correction for all tested confounders. In the general population, the associations between inflammatory biomarkers and rising levels of BP are mainly driven by age and fat mass. The stronger associations in women suggest that sex differences might exist in the complex interplay between BP and inflammation.

Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.

Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (≥140 mm Hg systolic blood pressure or  ≥90 mm Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention.

Free-breathing inner-volume black-blood imaging of the human heart using two-dimensionally selective local excitation at 3 T.

Black-blood fast spin-echo imaging is a powerful technique for the evaluation of cardiac anatomy. To avoid fold-over artifacts, using a sufficiently large field of view in phase-encoding direction is mandatory. The related oversampling affects scanning time and respiratory chest motion artifacts are commonly observed. The excitation of a volume that exclusively includes the heart without its surrounding structures may help to improve scan efficiency and minimize motion artifacts. Therefore, and by building on previously reported inner-volume approach, the combination of a black-blood fast spin-echo sequence with a two-dimensionally selective radiofrequency pulse is proposed for selective "local excitation" small field of view imaging of the heart. This local excitation technique has been developed, implemented, and tested in phantoms and in vivo. With this method, small field of view imaging of a user-specified region in the human thorax is feasible, scanning becomes more time efficient, motion artifacts can be minimized, and additional flexibility in the choice of imaging parameters can be exploited.

Obesity markers and blood pressure in a sample of Portuguese children and adolescents.

Little information exists regarding the effect of several obesity markers on blood pressure (BP) levels in youth. Transverse study including 2494 boys and 2589 girls. Height, weight and waist were measured according to the international criteria and body fat (BF) by bioimpedance. BP was measured by an automated device. Hypertension was defined using sex-specific, age-specific and height-specific observation-points. Body mass index (BMI) and waist were positively related with systolic blood pressure (SBP) and diastolic blood pressure (DBP) and heart rate in both sexes, whereas the relationships with BF were less consistent. Stepwise linear regression analysis showed that BMI was positively related with SBP and DBP in both sexes, whereas BF was negatively related with SBP in both sexes and with heart rate in boys only; finally, waist was positively related with SBP in boys and heart rate in girls. Age and heart rate-adjusted values of SBP and DBP increased with BMI: for SBP, 117+/-1, 123+/-1 and 124+/-1 mmHg in normal, overweight and obese boys, respectively; corresponding values for girls were 111+/-1, 114+/-1 and 116+/-2 mmHg (mean+/-SE, P<0.001). Overweight and obese boys had an odds ratio for being hypertensive of 2.26 (95% confidence interval: 1.79-2.86) and 3.36 (2.32-4.87), respectively; corresponding values for girls were 1.58 (confidence interval 1.25-1.99) and 2.31 (1.53-3.50). BMI, not BF or waist, is consistently and independently related to BP levels in children; overweight and obesity considerably increase the risk of hypertension.

Blood pressure loci identified with a gene-centric array.

Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 × 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery and follow-up data identified SNPs significantly associated with BP at p < 8.56 × 10(-7) at four further loci (NPR3, HFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.

Genetic dissection of sodium and potassium transport along the aldosterone-sensitive distal nephron: Importance in the control of blood pressure and hypertension.

In this review, we discuss genetic evidence supporting Guyton's hypothesis stating that blood pressure control is critically depending on fluid handling by the kidney. The review is focused on the genetic dissection of sodium and potassium transport in the distal nephron and the collecting duct that are the most important sites for the control of sodium and potassium balance by aldosterone and angiotensin II. Thanks to the study of Mendelian forms of hypertension and their corresponding transgenic mouse models, three main classes of diuretic receptors (furosemide, thiazide, amiloride) and the main components of the aldosterone- and angiotensin-dependent signaling pathways were molecularly identified over the past 20years. This will allow to design rational strategies for the treatment of hypertension and for the development of the next generation of diuretics.

Selective accumulation of differentiated FOXP3(+) CD4 (+) T cells in metastatic tumor lesions from melanoma patients compared to peripheral blood

Precise identification of regulatory T cells is crucial in the understanding of their role in human cancers. Here, we analyzed the frequency and phenotype of regulatory T cells (Tregs), in both healthy donors and melanoma patients, based on the expression of the transcription factor FOXP3, which, to date, is the most reliable marker for Tregs, at least in mice. We observed that FOXP3 expression is not confined to human CD25(+/high) CD4(+) T cells, and that these cells are not homogenously FOXP3(+). The circulating relative levels of FOXP3(+) CD4(+) T cells may fluctuate close to 2-fold over a short period of observation and are significantly higher in women than in men. Further, we showed that FOXP3(+) CD4(+) T cells are over-represented in peripheral blood of melanoma patients, as compared to healthy donors, and that they are even more enriched in tumor-infiltrated lymph nodes and at tumor sites, but not in normal lymph nodes. Interestingly, in melanoma patients, a significantly higher proportion of functional, antigen-experienced FOXP3(+) CD4(+) T was observed at tumor sites, compared to peripheral blood. Together, our data suggest that local accumulation and differentiation of Tregs is, at least in part, tumor-driven, and illustrate a reliable combination of markers for their monitoring in various clinical settings.

Correlations between imatinib pharmacokinetics, pharmacodynamics, adherence, and clinical response in advanced metastatic gastrointestinal stromal tumor (GIST): An emerging role for drug blood level testing?

Imatinib is the standard of care for patients with advanced metastatic gastrointestinal stromal tumors (GIST), and is also approved for adjuvant treatment in patients at substantial risk of relapse. Studies have shown that maximizing benefit from imatinib depends on long-term administration at recommended doses. Pharmacokinetic (PK) and pharmacodynamic factors, adherence, and drug-drug interactions can affect exposure to imatinib and impact clinical outcomes. This article reviews the relevance of these factors to imatinib's clinical activity and response in the context of what has been demonstrated in chronic myelogenous leukemia (CML), and in light of new data correlating imatinib exposure to response in patients with GIST. Because of the wide inter-patient variability in drug exposure with imatinib in both CML and GIST, blood level testing (BLT) may play a role in investigating instances of suboptimal response, unusually severe toxicities, drug-drug interactions, and suspected non-adherence. Published clinical data in CML and in GIST were considered, including data from a PK substudy of the B2222 trial correlating imatinib blood levels with clinical responses in patients with GIST. Imatinib trough plasma levels <1100ng/mL were associated with lower rates of objective response and faster development of progressive disease in patients with GIST. These findings have been supported by other analyses correlating free imatinib (unbound) levels with response. These results suggest a future application for imatinib BLT in predicting and optimizing therapeutic response. Nevertheless, early estimates of threshold imatinib blood levels must be confirmed prospectively in future studies and elaborated for different patient subgroups.

Remote control of pulmonary blood flow: a dream comes true.

The indication for pulmonary artery banding is currently limited by several factors. Previous attempts have failed to produce adjustable pulmonary artery banding with reliable external regulation. An implantable, telemetrically controlled, battery-free device (FloWatch) developed by EndoArt SA, a medical company established in Lausanne, Switzerland, for externally adjustable pulmonary artery banding was evaluated on minipigs and proved to be effective for up to 6 months. The first human implant was performed on a girl with complete atrioventricular septal defect with unbalanced ventricles, large patent ductus arteriosus and pulmonary hypertension. At one month of age she underwent closure of the patent ductus arteriosus and FloWatch implantation around the pulmonary artery through conventional left thoracotomy. The surgical procedure was rapid and uneventful. During the entire postoperative period bedside adjustments (narrowing or release of pulmonary artery banding with echocardiographic assessment) were repeatedly required to maintain an adequate pressure gradient. The early clinical results demonstrated the clinical benefits of unlimited external telemetric adjustments. The next step will be a multi-centre clinical trial to confirm the early results and adapt therapeutic strategies to this promising technology.

Obese and fit adolescents have lower blood pressure levels than obese and unfit counterparts.

The aim of the study was to assess the effects of physical fitness on the relationships between body mass index (BMI) and body fat (BF) on blood pressure (BP) levels. Cross-sectional study conducted in 25 schools of Lisbon (Portugal), including 2041 boys and 1995 girls aged 10-18. BF was assessed by bioimpedance. Cardiovascular fitness was assessed by the 20-meter shuttle run and classified as fit/unfit. Obesity (BMI or BF defined) was defined according to international criteria. In both sexes, BMI was positively related with systolic and diastolic BP, while BF was only positively related with diastolic BP z-scores. No interaction was found between fitness and BMI categories regarding BP levels, while for BF a significant interaction was found. Being fit reduced the BF-induced increase in the Odds ratio (OR) of presenting with high BP: OR (95% confidence interval) 1.01 (0.73-1.40) and 0.99 (0.70-1.38) for overweight and obese fit boys, respectively, the corresponding values for unfit overweight and obese boys being 1.33 (0.94-1.90) and 1.75 (1.34-2.28), respectively. The values were 0.88 (0.57-1.35) and 1.66 (0.98-2.80) for overweight and obese fit girls, respectively, the corresponding values for unfit overweight and obese being 1.63 (1.12-2.37) and 1.90 (1.32-2.73) respectively. No interaction was found between fitness and BMI-defined overweight and obesity. Being fit reduces the negative impact of BF on BP levels and high BP status in adolescents. This protective effect was not found with BMI.

Association between white-coat effect and blunted dipping of nocturnal blood pressure.

In this study, we assessed whether the white-coat effect (difference between office and daytime blood pressure (BP)) is associated with nondipping (absence of BP decrease at night). Data were available in 371 individuals of African descent from 74 families selected from a population-based hypertension register in the Seychelles Islands and in 295 Caucasian individuals randomly selected from a population-based study in Switzerland. We used standard multiple linear regression in the Swiss data and generalized estimating equations to account for familial correlations in the Seychelles data. The prevalence of systolic and diastolic nondipping (<10% nocturnal BP decrease) and white-coat hypertension (WCH) was respectively 51, 46, and 4% in blacks and 33, 37, and 7% in whites. When white coat effect and nocturnal dipping were taken as continuous variables (mm Hg), systolic (SBP) and diastolic BP (DBP) dipping were associated inversely and independently with white-coat effect (P < 0.05) in both populations. Analogously, the difference between office and daytime heart rate was inversely associated with the difference between daytime and night-time heart rate in the two populations. These results did not change after adjustment for potential confounders. The white-coat effect is associated with BP nondipping. The similar associations between office-daytime values and daytime-night-time values for both BP and heart rate suggest that the sympathetic nervous system might play a role. Our findings also further stress the interest, for clinicians, of assessing the presence of a white-coat effect as a means to further identify patients at increased cardiovascular risk and guide treatment accordingly.